Neospora for use in treating cancer and infectious diseases

ABSTRACT

The present invention relates to a strain of  Neospora caninum  for use in treating cancer or infectious diseases.

FIELD OF INVENTION

The present invention particularly relates to the use of at least onestrain of Neospora caninum or of a mutant thereof for treating cancer orinfectious diseases in a subject in need thereof.

BACKGROUND OF INVENTION

Despite considerable progress using surgery, chemotherapy, and radiationto treat cancer, the 5-year survival rate for many cancers is still verylow. An immune response directed to cancer cells may limit cancerdevelopment. However, tumor-mediated immunosuppression often blocksthese antitumor responses. Therefore, a novel option suggested fortreating cancer is to stimulate an effective immune response againsttumor cells.

In the art, the possibility of using microorganisms as powerfuladjuvants in the arsenal of anti-cancer immunotherapy has beenrecognized. Recent works highlighted the value of using attenuatedmicroorganisms including virus, bacteria and parasites for treatingcancer. For example, attenuated herpes virus expressing GM-CSFgenetically modified to target tumor cells has been approved by the FDAfor the treatment of metastatic melanomas (Amgen, IMLYGIC®). Moreover,attenuated microorganisms associated with another medicine are underclinical evaluation. For example, Opdivo® (nivolumab, IgG4 anti-PD1)associated to an attenuated bacterium Listeria monocytogenes is underclinical evaluation as an immunotherapy against non-small cell lungcancer. The combination of an attenuated virus modified to expressantigens E6 and E7 from the human papillomavirus HPV16 (TG4001) withavelumab (IgG1 anti-PD-L1) is evaluated in phase 1-2 in the treatment ofthe head and neck cancers positives for HPV. Listeria monocytogenes thatexpresses mesotheline (CRS-207) associated with pembrolizumab (IgG4anti-PD-1) is evaluated in phase 1 for the treatment of the gastriccancers.

Neospora caninum is an obligate intracellular protozoan parasiteresponsible for bovine neosporosis. Despite being taxonomically close toToxoplasma gondii, Neospora caninum presents significant differenceswith this protozoan parasite. In particular,

Neospora caninum does not infect humans The life cycle of Neosporacaninum is characterized by two distinct phases: (i) a sexual phase inthe final host (canids and dogs in particular) which leads to theproduction of oocysts, container of the sporozoites, eliminated indeposit and (ii) an asexual phase in an intermediate host (such as, forexample, sheep, goats, cattle, equidae, etc.) who leads to theproduction of tachyzoites, and then of cysts containing bradyzoites. Theinvasion process of the host cells by Neospora caninum comprises severalstages leading to the formation of a parasitophorous vacuole in whichthe parasite multiplies and develops. Active entry and vacuole formationresult from the coordinated secretion of parasite secretory organellesincluding rhoptries (ROP) and dense granules (GRA). The contents ofthese organelles are sequentially released during the lytic cycle andplay a crucial role in the host-parasite interactions.

The inventors surprisingly showed that the administration of at leastone strain of Neospora caninum to a subject induces a strong immuneresponse, in particular against tumors. The present invention thus aimsat providing a new treatment for cancer or infectious diseases based onthe use of Neospora caninum.

SUMMARY

The present invention relates to at least one strain of Neospora caninumfor use in treating cancer or an infectious disease. In one embodiment,the infectious disease is chronic. In one embodiment, said chronicinfectious disease is selected from chronic virus infection and chronicbacterial infection. In another embodiment, said chronic infectiousdisease is a chronic parasite infection.

In one embodiment, the strain of Neospora caninum is a wild type strain.

In another embodiment, the strain of Neospora caninum is a mutant straincharacterized by an over-expression of GRA15 protein, and/or by anunder-expression of ROP16 protein.

In one embodiment, the strain of Neospora caninum is at a tachyzoitestage.

In one embodiment, the strain of Neospora caninum is for use in treatingcancer, wherein cancer is a solid tumor, preferably an ovarian cancer, apancreatic cancer or a melanoma.

In another embodiment, the strain of Neospora caninum is for use intreating an infectious disease, preferably a chronic infectious disease,wherein said infectious disease, preferably said chronic infectiousdisease, is associated with or induces an immunodepletion orimmunosuppression, and is preferably selected from the group consistingof tuberculosis, HIV and malaria infections, preferably HIV ortuberculosis.

The present invention further relates to a composition for use intreating cancer or an infectious disease comprising the strain ofNeospora caninum in association with an excipient. In one embodiment,the infectious disease is chronic. In one embodiment, said chronicinfectious disease is selected from chronic virus infections and chronicbacterial infections. In another embodiment, said chronic infectiousdisease is a chronic parasite infection.

In one embodiment, the composition is a pharmaceutical composition andfurther comprises at least one pharmaceutically acceptable excipient.

In one embodiment, the composition is a vaccine composition for use intreating cancer or an infectious disease comprising the at least onestrain of Neospora caninum, wherein preferably the vaccine compositioncomprises an adjuvant. In one embodiment, the infectious disease ischronic. In one embodiment, said chronic infectious disease is selectedfrom chronic virus infections and chronic bacterial infections. Inanother embodiment, said chronic infectious disease is a chronicparasite infection.

In one embodiment, the strain of Neospora caninum for use, thecomposition for use or the vaccine composition for use as described inthe present invention is to be administered to the subject viasubcutaneous, intradermal or intratumoral routes.

In one embodiment, the amount of strains of Neospora caninum to beadministered to the subject is ranging from about 10⁴ to about 10¹¹.

The present invention further relates to a mutant strain of Neosporacaninum characterized by an over-expression of GRA15 protein, and/or anunder-expression of ROP16 protein.

The present invention also relates to a composition comprising themutant strain of Neospora caninum.

The present invention further relates to a pharmaceutical compositioncomprising the mutant strain of Neospora caninum in association with atleast one pharmaceutically acceptable excipient.

The present invention relates to a vaccine composition comprising themutant strain of Neospora caninum, wherein preferably the vaccinecomposition comprises an adjuvant.

The present invention further relates to an ex vivo method foractivating T cells, comprising contacting T cells with a strain ofNeospora caninum.

Definitions

In the present invention, the following terms have the followingmeanings:

-   -   “Treatment” refers to both therapeutic treatment and        prophylactic or preventative measures; wherein the object is to        prevent or slow down (lessen) the targeted pathologic condition        or disorder. Those in need of treatment include those already        with the disorder as well as those prone to have the disorder or        those in whom the disorder is to be prevented. A subject is        successfully “treated” for the targeted pathologic condition or        disorder if, after receiving a therapeutic amount of a strain of        Neospora caninum as described herein, the subject shows        observable and/or measurable improvement in one or more of the        following: reduction in the number of pathogenic cells;        reduction in the percent of total cells that are pathogenic;        relief to some extent of one or more of the symptoms associated        with the targeted pathologic condition or disorder; reduced        morbidity and mortality, and/or improvement in quality of life        issues. The above parameters for assessing successful treatment        and improvement in the disease are readily measurable by routine        procedures familiar to a physician.    -   “Therapeutically effective amount” refers to level or amount of        agent that is aimed at, without causing significant negative or        adverse side effects to the target, (1) delaying or preventing        the onset of the targeted pathologic condition or disorder; (2)        slowing down or stopping the progression, aggravation, or        deterioration of one or more symptoms of the targeted pathologic        condition or disorder; (3) bringing about ameliorations of the        symptoms of the targeted pathologic condition or disorder; (4)        reducing the severity or incidence of the targeted pathologic        condition or disorder; (5) curing the targeted pathologic        condition or disorder. An effective amount may be administered        prior to the onset of the targeted pathologic condition or        disorder, for a prophylactic or preventive action. Alternatively        or additionally, the effective amount may be administered after        initiation of the targeted pathologic condition or disorder, for        a therapeutic action.    -   “Pharmaceutically acceptable excipient” refers to an excipient        that does not produce an adverse, allergic or other untoward        reaction when administered to an animal, preferably a human. It        includes any and all dispersion media and solvents, coatings,        isotonic and absorption delaying agents, additives,        preservatives, stabilizers and the like. For human        administration, preparations should meet sterility,        pyrogenicity, general safety and purity standards as required by        regulatory offices, such as, for example, FDA Office or EMA.    -   “Subject” refers to a mammal, preferably a human. In one        embodiment, a subject may be a “patient”, i.e., a warm-blooded        animal, more preferably a human, who/which is awaiting the        receipt of, or is receiving medical care or was/is/will be the        object of a medical procedure, or is monitored for the        development of a disease.

In one embodiment, the subject is an adult (for example a subject abovethe age of 18). In another embodiment, the subject is a child (forexample a subject below the age of 18). In one embodiment, the subjectis a male. In another embodiment, the subject is a female.

-   -   “About” preceding a figure means plus or less 10% of the value        of said figure.    -   “Vaccine” refers to any preparation comprising substance or        group of substances meant to induce an immune response in a        subject, e.g., against a cancer cell, a tumor or against cells        infected with an intracellular pathogen, such as, for example,        Mycobacterium tuberculosis, HIV or plasmodium infected cells. As        used herein, the term “vaccine” refers both to prophylactic        vaccines and to therapeutic vaccines. Prophylactic vaccines are        used to prevent a subject from the occurrence of a disease or        condition (e.g., cancer or an infectious disease), or to limit        the severity of the disease or condition, such that the subject        administered with the vaccine only develops mild symptoms of the        disease or condition. Therapeutic vaccines are intended to treat        the targeted disease or condition, e.g., cancer or an infection        disease, such as, for example, tuberculosis, HIV or malaria        infections in a subject.    -   “Immunodepletion” or “immunosuppression” refer to a deficient        immune system, i.e., an immune system for which one or more cell        lines are either absent or deficient.    -   “Activated cells” refers to the state of an immune cell, in        particular a T cell, that has been sufficiently stimulated to        induce a detectable cellular response. Activation can also be        associated with detectable effector function(s) such as cytokine        production or suppressive activity.    -   “Autologous” refers to any material derived from the same        individual to whom it is later to be re-introduced.    -   “Allogeneic” refers to any material derived from a different        individual of the same specie as the individual to whom the        material is introduced. Two or more individuals are said to be        allogeneic to one another when the genes at one or more loci are        not identical. In some aspects, allogeneic material from        individuals of the same species may be sufficiently unlike        genetically to interact antigenically.    -   “Substantially purified” refers to a cell (e.g., a strain        Neospora caninum) that is essentially free of other cell types        or organisms (e.g., of other protozoan organisms). In one        embodiment, a substantially purified strain refers to a strain        which is at least about 75% free, 80% free, or 85% free, and        preferably about 90%, 95%, 96%, 97%, 98%, or 99% free, from        other cell types or organisms.

DETAILED DESCRIPTION

The present invention relates to a strain of Neospora caninum for use intreating cancer or an infectious disease in a subject in need thereof.

Indeed, the inventors have demonstrated that the administration of astrain of Neospora caninum can induce an immune response, for example,against a tumor or against cells infected with an intracellularpathogen, thereby allowing the treatment of the disease or condition.

Neospora caninum presents the advantage of being noninfectious in human.Moreover, Neospora caninum presents no risk of encystment that couldlead to unpredictable side effects after several years of treatment.

Several strains of Neospora caninum have been described and are wellknown in the art. Examples of strains of Neospora caninum that may beused in the present invention include, but are not limited to, Neosporacaninum 1 (NC-1), Neospora caninum Liverpool, BPA1, BPA6, NC-Beef,NC-Illinois, NC-LivB1, NC-LivB2, NC-SweB1, JAP1, NC-GER1, NC-GER2,NC-GER 3, NC-GER 4, NC-GER 5, NC-GER6, NC-GER8, NC-GER9, NC-Bahia,NC-Nowra, WA-K9, NcNZ1, NcNZ2, NcNZ3 and NcIs491. In one embodiment, thestrain of Neospora caninum is Neospora caninum 1 (NC-1).

In one embodiment, the strain of Neospora caninum is a wild type strain.

In another embodiment, the strain of Neospora caninum is a mutantstrain.

As used herein, a “mutant” strain is intended to mean a straincomprising at least one mutation as compared to the wild type strainfrom which it derives, wherein the term “mutation” may refer, withoutlimitation, to point mutations, insertions, deletions, amplifications orgene duplications.

According to one embodiment of the invention, the mutant straincomprises a genome having at least about 70% identity with the genome ofthe wild type Neospora caninum strain from which it derives, preferablyat least about 80%, 90%, 95%, 96%, 97%, 98% or 99% of identity.

Within the meaning of the present invention, the term “identity”, whenit is used in a relationship between the sequences of two or morenucleotide sequences, refers to the degree of relationship between thesenucleotide sequences, as determined by the number of correspondencesbetween the chains of two bases or more. According to the invention,“identity” corresponds to a percentage of identity between two sequencesor more. This percentage is defined as a number of positions for whichthe bases are identical when the sequences are aligned optimally,divided by the total number of bases of the smaller of the twosequences. The differences between the sequences may be distributed atrandom and over all their lengths. Two sequences are said to be alignedoptimally when the percentage of identity is maximal. Moreover, as willbe clear to a person skilled in the art, it may be necessary to haverecourse to additions of gaps so as to obtain an optimum alignmentbetween the two sequences. The percentage of identity between twonucleic acid sequences can therefore be determined by comparing thesetwo sequences aligned optimally in which the nucleic acid sequence to becompared may comprise additions or deletions compared with the referencesequence for optimum alignment between these two sequences. Thepercentage of identity is then calculated by determining the number ofidentical positions for which the nucleotide is identical between thetwo sequences, dividing this number of identical positions by the totalnumber of positions in the comparison window and multiplying the resultobtained by 100 in order to obtain the percentage identity between thesetwo sequences. Preferably, the methods for determining identity aredesigned to give the greatest possible agreement between the comparedsequences. The percentage identity can be determined by a particularmathematical model or by a computer program (generally designated by theterm “algorithm”). Methods for calculating the identity betweennucleotide sequences are well known to persons skilled in the art.Non-limitative examples of such methods include those described in thefollowing documents: Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing:

Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data Part 1, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987;Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M.Stockton Press, New York, 1991; and Carillo et al., SIAM J. AppliedMath., 48, 1073 (1988). Methods for determining identity have beendescribed in computer programs accessible to the public. Preferredexamples of methods using computer programs include, without beinglimited thereto, the GCG software, including GAP (Devereux et al., Nucl.Acid. Res. \2, 387 (1984); Genetics Computer Group, University ofWisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al.,J. Mol. Biol. 215, 403-410 (1990)). The BLASTX program is available fromthe National Center for Biotechnology Information (NCBI) and othersources (BLAST Manual, Altschul et al., NCB/NLM/NIH Bethesda, Md. 20894;Altschul et al., supra). The Smith-Waterman algorithm, which is wellknown to persons skilled in the art, can also be used to determine thepercentage identity between two sequences.

In one embodiment, the mutant strain of Neospora caninum over-expressesGRA15.

As used herein, the term “GRA-15 protein” refers to “Dense GranuleProtein 15” of Neospora caninum. The GRA15 protein is stored insecretory compartments of Neospora caninum (called dense granules) andsecreted into the vacuole after host cell invasion. In one embodiment,GRA15 refers to the homologue in Neospora caninum of the GRA15 gene ofToxoplasma gondii (having the accession number: Gene ID: 7895856, locustag TGME49_275470).

As used herein, the term “over-expression” refers to an increasedexpression level as compared to a reference expression level, such as,for example, an expression level superior or equal to 110%, 120%, 130%,140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500%, 600%, 700%,800% 900% or 1000% or more of the reference expression level.

Methods to obtain a cell over-expressing a protein are well known in theart. For example, an over-expression of a protein may be obtained byconstructing a “knock-in” of the gene encoding this protein. Examples ofmethods for constructing a knock-in include, but are not limited to,electroporation, lipofection, calcium phosphate transfection and thelike.

As used herein, the term “expression level” can refer alternatively tothe transcription level of a gene (i.e., expression is measured at theRNA level) or to the translation level of the gene (i.e., expression ismeasured at the protein level).

Methods for assessing the transcription level of a gene are well knownin the prior art. Examples of such methods include, but are not limitedto, RT-PCR, RT-qPCR, Northern Blot, hybridization techniques such as,for example, use of microarrays, and combination thereof including butnot limited to, hybridization of amplicons obtained by RT-PCR,sequencing such as, for example, next-generation DNA sequencing (NGS) orRNA-seq (also known as “Whole Transcriptome Shotgun Sequencing”) and thelike.

Methods for determining a protein level in a sample are well-known inthe art. Examples of such methods include, but are not limited to,immunohistochemistry, Multiplex methods (Luminex), Western blot,enzyme-linked immunosorbent assay (ELISA), sandwich ELISA,fluorescent-linked immunosorbent assay (FLISA), enzyme immunoassay(EIA), radioimmunoassay (RIA), flow cytometry (FACS) and the like.

In one embodiment, the reference expression level (which may correspondto a reference protein expression level or a reference mRNA expressionlevel as hereinabove described) corresponds to the expression levelmeasured in a reference Neospora caninum strain, preferably in a wildtype Neospora caninum strain.

In another embodiment, the mutant strain of Neospora caninumunderexpresses ROP16, preferably the mutant strain does not expressROP16 protein. For example, the mutant strain of Neospora caninum is aknockout mutant for the gene encoding for ROP16 proteins)(ROP16^(KO)).

As used herein, the term “ROP16 protein” refers to “Rhoptry Protein 16”.In one embodiment, ROP16 refers to the homologue in Neospora caninum ofthe ROP16 gene of Toxoplasma gondii (having the accession number: GeneID: 7894782).

As used herein, the term “under-expression” (or down-expression) refersto a decreased expression level as compared to a reference expressionlevel, such as, for example, an expression level inferior or equal to90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less of the referenceexpression level, preferably of the expression level measured in a wildtype Neospora caninum strain.

As used herein, the term “knockout for the gene” denotes a geneticmutation resulting in a loss of function and/or a loss of expression ofthe protein encoded by the said gene. In one embodiment, said geneticmutation corresponds to the disruption of all or a portion of a gene ofinterest, preferably the total disruption of the gene. Preferably, thedeletion starts at or before the start codon of the deleted gene, andends at or after the stop codon of the deleted gene. Other examples ofgenetic mutations include, but are not limited to, substitution,deletion, or insertion.

In one embodiment, the knockout of ROP16 protein can be achieved byreplacing the coding sequence with a nucleic acid molecule encoding aselectable marker, replacing the coding sequence with a nucleic acidmolecule encoding an exogenous protein, substituting the promoter with amutated promoter which can no longer be recognized by Neospora caninumtranscription proteins (i.e., a promoter mutation), etc.

In one embodiment, the mutant strain of Neospora caninum over-expressesGRA15 and under-expresses ROP16.

In one embodiment, the strain of Neospora caninum is isolated, inparticular is isolated from its natural environment.

In one embodiment, the strain of Neospora caninum is substantiallypurified.

In one embodiment, a living strain of Neospora caninum is used in thepresent invention.

In one embodiment, the strain of Neospora caninum is a tachyzoite. By“tachyzoite” is meant the rapidly multiplying form of Neospora caninum.The tachyzoite has usually a crescent shape and a variable size, forexample a size of about 5-8×2-3 μm. The apical part of the parasitecomprises conoids which participate in the penetration of the parasiteinto the host cell. The micronemes, the rhoptries and the dense granulesconstitute the three major organelles of the tachyzoite, which alsocomprises a nucleus, an apicoplast, a Golgi apparatus, an endoplasmicreticulum and an organite similar to the mitochondrion.

In one embodiment, the strain of Neospora caninum is a tachyzoite fromNeospora caninum-1 (NC-1).

Methods for obtaining and maintaining living strains, in particulartachyzoites, of Neospora caninum are well known by the skilled artisan.An example of a method for maintaining strains of Neospora caninum isindicated below.

Tachyzoites of the NC-1 strain of Neospora caninum are harvested frominfected human foreskin fibroblasts (such as, for example, Hs 27 (ATCCCRL-1634) cultured in monolayers in DMEM, supplemented with 10%heat-inactivated FCS, 50 U/ml penicillin/50 μg/ml streptomycin, and 1%HEPES). Neospora caninum tachyzoites may be harvested when monolayers ofhuman foreskin fibroblasts are completely lysed.

In one embodiment, Neospora caninum is for treating cancer.

In one embodiment, Neospora caninum is for treating a tumor, preferablya solid tumor. Indeed, the Inventors have shown that the administrationof Neospora caninum induces a decrease of the tumor volume in mice (seeExamples). Therefore, in one embodiment, Neospora caninum is forinducing a decrease of the tumor volume and/or for preventing anincrease of the tumor volume in a subject in need thereof.

In one embodiment, the cancer may be any solid tumor. Examples of solidtumors include but are not limited to, bile duct cancer (e.g.,periphilar cancer, distal bile duct cancer, intrahepatic bile ductcancer), bladder cancer, bone cancer (e.g., osteoblastoma,osteochrondroma, hemangioma, chondromyxoid fibroma, osteosarcoma,chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant celltumor of the bone, chordoma, lymphoma, multiple myeloma), brain andcentral nervous system cancer (e.g., meningioma, astocytoma,oligodendrogliomas, ependymoma, gliomas, medulloblastoma, ganglioglioma,Schwannoma, germinoma, craniopharyngioma), breast cancer (e.g., ductalcarcinoma in situ, infiltrating ductal carcinoma, infiltrating lobularcarcinoma, lobular carcinoma in, situ, gynecomastia), Castleman disease(e.g., giant lymph node hyperplasia, angio follicular lymph nodehyperplasia), cervical cancer, colorectal cancer, endometrial cancer(e.g., endometrial adenocarcinoma, adenocanthoma, papillary serousadnocarcinroma, clear cell), esophagus cancer, gallbladder cancer (e.g.,mucinous adenocarcinoma, small cell carcinoma), gastrointestinalcarcinoid tumors (e.g., choriocarcinoma, chorioadenoma destruens),Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidneycancer (e.g., renal cell cancer), laryngeal and hypopharyngeal cancer,liver cancer (e.g., hemangioma, hepatic adenoma, focal nodularhyperplasia, hepatocellular carcinoma), lung cancer (e.g., small celllung cancer, non-small cell lung cancer), mesothelioma, plasmacytoma,nasal cavity and paranasal sinus cancer (e.g., esthesioneuroblastoma,midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavityand oropharyngeal cancer, ovarian cancer, pancreatic cancer, penilecancer, pituitary cancer, prostate cancer, retinoblastoma,rhabdomyosarcoma (e.g., embryonal rhabdomyosarcoma, alveolarrhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer,skin cancer (e.g., melanoma, nonmelanoma skin cancer), stomach cancer,testicular cancer (e.g., seminoma, nonseminoma germ cell cancer), thymuscancer, thyroid cancer (e.g., follicular carcinoma, anaplasticcarcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma,thyroid lymphoma), vaginal cancer, vulvar cancer, and uterine cancer(e.g., uterine leiomyosarcoma).

In one embodiment, the cancer is selected from ovarian cancer,pancreatic cancer and melanoma.

In one embodiment, the cancer is glioma.

In one embodiment, Neospora caninum is for treating metastatic cancer.In one embodiment, Neospora caninum is for preventing the occurrence ofmetastasis, and/or for reducing the number of metastasis in a subject inneed thereof.

In one embodiment, Neospora caninum is for treating blood cancer. In oneembodiment, Neospora caninum is for treating an infectious disease.

In one embodiment, said infectious disease is a chronic infectiousdisease, i.e., a disease due to the prolonged and persistent invasion ofthe body of a subject by pathogens (including, for example, parasites,bacteria (in particular mycobacteria) and viruses).

In one embodiment, said infectious disease is a chronic virus infection,such as, for example, a HIV infection.

In one embodiment, said infectious disease is a chronic bacterialinfection, such as, for example, an infection by Mycobacteriumtuberculosis.

In one embodiment, said infectious disease is a chronic infection with aparasite, such as, for example, an infection by plasmodium.

In one embodiment, said chronic infectious disease is caused by thepresence of an intracellular pathogen, such as, for example, anintracellular virus, an intracellular bacterium or an intracellularparasite.

In one embodiment, said infectious disease is a chronic infectiousdisease associated with an immunodepletion or an immunosuppression orinducing an immunodepletion or an immunosuppression.

Examples of chronic infectious diseases associated with or inducing animmunodepletion or an immunosuppression include, but are not limited to,tuberculosis, HIV or malaria infections.

In one embodiment, said infectious disease is not due to an apicomplexanof the family Cryptosporidiidae. In one embodiment, said infectiousdisease is not a cryptosporidiosis. In one embodiment, said infectiousdisease is not neosporosis.

In one embodiment, the subject is a human.

In one embodiment, the subject is immunosuppressed, i.e., presents animpaired immune system. In one embodiment, the immune system of thesubject has compromised ability to fight a cancer or an infectiousdisease.

In one embodiment, the subject has cancer. In one embodiment, thesubject is diagnosed or has been diagnosed with cancer.

In one embodiment, the cancer is early or late stage cancer.

In one embodiment, the subject was not treated previously with anothertreatment for cancer (i.e., the method of the invention is the firstline treatment).

In another embodiment, the subject previously received one, two or moreother treatments for cancer (i.e., the method of the invention is asecond line, a third line or more). In one embodiment, the subjectpreviously received one or more other treatments for cancer, but wasunresponsive or did not respond adequately to these treatments, whichmeans that there is no or too low therapeutic benefit induced by thesetreatments.

In another embodiment, the subject is at risk of developing cancer.Examples of risk factors for developing cancer include, but are notlimited to, family history of cancer, genetic predisposition, orexposure to a carcinogen.

In another embodiment, the subject has an infectious disease, preferablya chronic infectious disease. In one embodiment, the subject has aninfectious disease (preferably a chronic infectious disease) associatedwith or inducing an immunodepletion or an immunosuppression. In oneembodiment, the subject has a chronic infectious disease caused by anintracellular pathogen. In one embodiment, the subject has atuberculosis infection. In another embodiment, the subject has an HIVinfection. In another embodiment, the subject has a malaria infection.

In one embodiment, the subject was not treated previously with anothertreatment for the said infection (i.e., the method of the invention isthe first line treatment).

In another embodiment, the subject previously received one, two or moreother treatments for the said infection (i.e., the method of theinvention is a second line, a third line or more). In anotherembodiment, the subject previously received one or more other treatmentsfor the said infection, but was unresponsive or did not respondadequately to these treatments, which means that there is no or too lowtherapeutic benefit induced by these treatments.

In one embodiment, the subject is not affected by a cryptosporidiosis.

The present invention further relates to a composition for use intreating cancer or infectious diseases in a subject in need thereofcomprising, consisting or consisting essentially of at least one strainof Neospora caninum or a mutant thereof as described hereinabove.

As used herein, “consisting essentially of”, with reference to acomposition, means that at least one strain of Neospora caninum or amutant thereof of the invention is the only one therapeutic agent oragent with a biologic activity within said composition.

Another object of the invention is a pharmaceutical composition for usein treating cancer or infectious diseases in a subject in need thereofcomprising, consisting or consisting essentially of at least one strainof Neospora caninum or a mutant thereof as described hereinabove and atleast one pharmaceutically acceptable excipient.

Examples of pharmaceutically acceptable excipients include, but are notlimited to, media, solvents, coatings, isotonic and absorption delayingagents, additives, stabilizers, preservatives, surfactants, substanceswhich inhibit enzymatic degradation, alcohols, pH controlling agents,and propellants.

Examples of pharmaceutically acceptable media include, but are notlimited to, water, phosphate buffered saline, normal saline or otherphysiologically buffered saline, or other solvent such as glycol,glycerol, and oil such as olive oil or an injectable organic ester. Apharmaceutically acceptable medium can also contain liposomes ormicelles, and can contain immunostimulating complexes prepared by mixingpolypeptide or peptide antigens with detergent and a glycoside.

Examples of coating materials include, but are not limited to, lecithin.

Examples of isotonic agents include, but are not limited to, sugars,sodium chloride, and the like. Examples of agents that delay absorptioninclude, but are not limited to, aluminum monostearate and gelatin.

Examples of additives include, but are not limited to, mannitol,dextran, sugar, glycine, lactose or polyvinylpyrrolidone or otheradditives such as antioxidants or inert gas, stabilizers or recombinantproteins (e.g., human serum albumin) suitable for in vivoadministration.

Examples of suitable stabilizers include, but are not limited to,sucrose, gelatin, peptone, digested protein extracts such as NZ-Amine orNZ-Amine AS.

Another object of the invention is a medicament for use in treatingcancer or infectious diseases in a subject in need thereof comprising,consisting or consisting essentially of at least one strain of Neosporacaninum or a mutant thereof as described herein.

Another object of the invention is a vaccine composition for use intreating cancer or infectious diseases in a subject in need thereofcomprising, consisting or consisting essentially of at least one strainof Neospora caninum or a mutant thereof as described herein. In oneembodiment, the vaccine of the invention is a prophylactic vaccine. Inanother embodiment, the vaccine of the invention is a therapeuticvaccine.

In one embodiment, the vaccine composition further comprises at leastone adjuvant.

As used herein, the term “adjuvant” refers to a substance that enhances,augments or potentiates the host's immune response induced by the strainof Neospora caninum.

Examples of adjuvants that can be used in the vaccine compositioninclude, but are not limited to, ISA51; emulsions such as CFA, MF59,montanide, AS03 and AF03; mineral salts such as alum, calcium phosphate,iron salt, zirconium salt, and AS04; TLR ligands such as TLR2 ligands(such as outer-surface protein A or OspA), TLR3 ligands (such as polyI:C), TLR4 ligands (such as MPL and GLA), TLR5 ligands, TLR7/8 ligands(such as imiquimod), TLR9 ligands (such as CpG ODN); polysacharridessuch as chitin, chitosan, α-glucans, β-glucans, fructans, mannans,dextrans, lentinans, inulin-based adjuvants (such as gamma inulin); TLR9and STING ligands such as K3 CpG and cGAMP.

The strain of Neospora caninum, the composition, the pharmaceuticalcomposition, the medicament or the vaccine composition of the presentinvention may be administered orally, parenterally, by intraperitonealadministration, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir.

In one embodiment, the strain of Neospora caninum, the composition, thepharmaceutical composition, the medicament or the vaccine composition ofthe present invention is injected. Examples of injections include, butare not limited to, intratumoral, intradermal, subcutaneous,intravenous, intramuscular, intra-lymphatic, intra-articular,intra-synovial, intrasternal, intrathecal, intravesical, intrahepatic,intralesional and intracranial injection or infusion techniques.

In one embodiment, the strain of Neospora caninum is administeredsubcutaneously. In one embodiment, the strain of Neospora caninum isadministered intradermally. In one embodiment, the strain of Neosporacaninum is administered intratumorally.

In one embodiment, the strain, the composition, the pharmaceuticalcomposition, the medicament or the vaccine composition of the presentinvention is in a form adapted to oral administration. According to afirst embodiment, the form adapted to oral administration is a solidform selected from the group comprising tablets, pills, capsules, softgelatin capsules, sugarcoated pills, orodispersing tablets, effervescenttablets or other solids. According to a second embodiment, the formadapted to oral administration is a liquid form, such as, for example, adrinkable solution, a buccal spray, liposomal forms and the like.

In another embodiment, the strain, the composition, the pharmaceuticalcomposition, the medicament or the vaccine composition of the presentinvention is formulated for rectal or vaginal administration and may bepresented as suppositories, pessaries, tampons, creams, gels, pastes,foams or sprays.

In another embodiment, the strain, the composition, the pharmaceuticalcomposition, the medicament or the vaccine composition of this inventionis in a form suitable for parenteral administration. Forms suitable forparenteral administrations include, but are not limited to, sterileisotonic aqueous or non-aqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use.

In another embodiment, the strain, the composition, the pharmaceuticalcomposition, the medicament or the vaccine composition of the inventionis in a form adapted for local delivery via the nasal and respiratoryroutes. Examples of formulations suitable for nasal or respiratoryadministration include, but are not limited to, nasal solutions, sprays,aerosols and inhalants.

In another embodiment, the strain, the composition, the pharmaceuticalcomposition, the medicament or the vaccine composition of the inventionis in a form adapted to a topical administration. Examples offormulations adapted to a topical administration include, but are notlimited to, ointment, paste, eye drops, cream, patch, such as, forexample, transdermal patch, gel, liposomal forms and the like.

In one embodiment, the composition or formulation of the invention maybe presented in unit-dose or multi-dose sealed containers, for example,ampoules and vials, and may be stored in a lyophilized conditionrequiring only the addition of the sterile liquid excipient, for examplewater for injection, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets.

The exact dose for administration can be determined by the skilledpractitioner, in light of factors related to the subject that requirestreatment. Dosage is adjusted to provide sufficient levels of thecomposition or to maintain the desired effect of reducing signs orsymptoms of the targeted pathologic condition or disorder, or reducingseverity of the targeted pathologic condition or disorder. Factors whichmay be taken into account include the severity of the disease state(such as for example the tumor volume or the number of infected cells),the prognosis of the disease, the localization or accessibility to thetumor, general health of the subject, age, weight, and gender of thesubject, diet, time and frequency of administration, drugcombination(s), reaction sensitivities, and tolerance/response totherapy.

In one embodiment, a therapeutically effective amount of the strain, thecomposition, the pharmaceutical composition, the medicament or thevaccine composition of the present invention is administered (or is tobe administered) to the subject.

In one embodiment of the invention, the strain of Neospora caninum orthe composition, the pharmaceutical composition, the medicament or thevaccine composition is administered at least once, preferably at leasttwice, more preferably at least three times, and even more preferably atleast four times at day, week or month intervals, according to aprime/boost mode.

In one embodiment of the invention, the amount of strains of Neosporacaninum (preferably of tachyzoites) as described hereinaboveadministered per administration ranges from about 10⁴ to about 10¹¹,preferably from about 10⁵ to about 10¹⁰, more preferably from about 10⁶to about 10⁹, and even more preferably from about 10⁷ to about 10⁸.

In one embodiment of the invention, the daily amount of strains ofNeospora caninum (preferably of tachyzoites) as described hereinaboveadministered per day ranges from about 10⁴ to about 10¹¹ per day,preferably from about 10⁵ to about 10¹⁰/day, more preferably from about10⁶ to about 10⁹/day, and even more preferably from about 10⁷ to about10⁸/day.

The present invention further relates to a method for treating cancer oran infectious disease in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of astrain of Neospora caninum or mutant thereof.

In one embodiment, the method of the invention is a method fordecreasing tumor volume, and/or for preventing any increase of tumorvolume.

In one embodiment, the method of the invention is for preventing theoccurrence of metastasis, and/or for reducing the number of metastasis.

The present invention relates to a method for inducing an immuneresponse against a cancer cell, a tumor or an infected cell, preferablya cell infected with an intracellular pathogen, such as, for example,Mycobacterium tuberculosis, HIV or plasmodium in a subject in needthereof. In one embodiment, the immune response is a non-specificresponse. In one embodiment, the immune response is characterized by thesecretion of IFNγ.

As used herein, “an immune response” is a detectable immune responsee.g., T cell immune response or antibody production. Methods formeasuring a T cell immune response are well known by the skilled artisanand include, without limitation, monitoring the production of IFN-gamma.

The Inventors show herein that the administration to a subject ofstrains of Neospora caninum is able to induce cellular and humoralimmune responses. Indeed, the Inventors show a four-fold increasesecretion of IFNgamma after Neospora caninum administration and specificIgG against Neospora caninum are detected in the serum after Neosporacaninum administration (see Examples).

The present invention thus relates to a method for inducing a cellularand/or humoral immune response in a subject, wherein said methodcomprises administering to the subject at least one cell of the strainof Neospora caninum to the subject.

The present invention relates to a method for inducing a decrease of thetumor volume, preferably a decrease of the tumor volume of at least 25%,preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, whereinsaid method comprises administering to the subject at least one cell ofthe strain of Neospora caninum to the subject.

Indeed, the Inventors show herein that the administration of strains ofNeospora caninum leads to the decrease of the tumor volume. Morespecifically, a 3-fold decrease of the tumor volume is observed after aperitumoral administration (see Examples).

The present invention further relates to an ex vivo method foractivating cells, comprising contacting cells, preferably human cells,more preferably PBMC (peripheral blood mononuclear cells) and even morepreferably T cells, with a strain of Neospora caninum as described inthe present invention.

The present invention thus further relates to the use of a strain ofNeospora caninum as described in the present invention for ex vivoactivating cells, preferably human cells, more preferably PBMC and evenmore preferably T cells, wherein said cells are contacted with saidstrain of Neospora caninum.

In one embodiment, said cells are recovered from a patient with canceror with an infectious disease. In one embodiment, the step of recoveringthe cells from a patient is not part of the method of the invention.

In one embodiment, the activated cells are to be administered to apatient with cancer or with an infectious disease, thereby treatingcancer or an infectious disease. In one embodiment, the activated cellsare autologous to the cells of the patient. In another embodiment, theactivated cells are allogenic to the cells of the patient.

The present invention further relates to a method for treating cancer oran infectious disease in a subject in need thereof, comprising:

-   -   contacting cells, preferably PBMC, more preferably T cells with        a strain of Neospora caninum as described in the present        invention, wherein preferably said cells are previously        recovered from said subject, and    -   administering the activated cells to the subject, thereby        treating cancer or an infectious disease.

Another object of the invention is a mutant strain of Neospora caninum,wherein said mutant strain overexpresses GRA15 and/or under-expressesROP16.

Examples of strains of Neospora caninum from which the mutant strain mayderive include, but are not limited to, Neospora caninum 1 (NC-1),Neospora caninum Liverpool, BPA1, BPA6, NC-Beef, NC-Illinois, NC-LivB1,NC-LivB2, NC-SweB1, JAP1, NC-GER1, NC-GER2, NC-GER 3, NC-GER 4, NC-GER5, NC-GER6, NC-GER8, NC-GER9, NC-Bahia, NC-Nowra, WA-K9, NcNZ1, NcNZ2,NcNZ3 and NcIs491. In one embodiment, the mutant strain of Neosporacaninum derives from wild type Neospora caninum-1 (NC-1). In anotherembodiment, the mutant strain of Neospora caninum derives from wild typeNeospora caninum Liverpool.

In one embodiment of the invention, the mutant strain comprises a genomehaving more than 70% identity with the genome of the wild type strain ofNeospora caninum from which it derives, preferably more than 80%, 90%,95%, 96%, 97%, 98% or 99% of identity.

In one embodiment, the mutant strain of Neospora caninum over-expressesGRA15.

In another embodiment, the mutant strain of Neospora caninumunder-expresses ROP16, preferably the mutant strain does not expressROP16 protein. For example, the mutant strain of Neospora caninum is aknockout mutant for the gene encoding for ROP16 proteins)(ROP16^(KO)).

In one embodiment, the knockout of ROP16 protein can be achieved byreplacing the coding sequence with a nucleic acid molecule encoding aselectable marker, replacing the coding sequence with a nucleic acidmolecule encoding an exogenous protein, substituting the promoter with amutated promoter which can no longer be recognized by Neospora caninumtranscription proteins (i.e., a promoter mutation), etc.

In one embodiment, the mutant strain of Neospora caninum over-expressesGRA15 and under-expresses ROP16.

Another object of the present invention is a composition comprising,consisting or consisting essentially of at least one mutant strain ofNeospora caninum as described hereinabove.

Another object of the invention is a pharmaceutical compositioncomprising, consisting or consisting essentially of at least one mutantstrain of Neospora caninum as described hereinabove and at least onepharmaceutically acceptable excipient.

Examples of pharmaceutically acceptable excipients are described herein.

Another object of the invention is a medicament comprising, consistingor consisting essentially of at least one mutant strain of Neosporacaninum as described hereinabove.

Another object of the invention is a vaccine composition comprising,consisting or consisting essentially of at least one mutant strain ofNeospora caninum as described hereinabove.

In one embodiment, the vaccine composition further comprises at leastone adjuvant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a dose-response curve showing the percent survival of miceafter the administration of different doses of wild type strains ofNeospora caninum-1 (NC-1).

FIG. 2 is a histogram showing the seric IFNgamma production at day 2 orday 8 after administration of different doses of wild type strains ofNeospora caninum-1 (NC-1) in mice.

FIG. 3 is a dot plot showing the production of specific IgG againstNeospora caninum after administration of different doses of wild typestrains of Neospora caninum-1 (NC-1) in mice.

FIG. 4 is a curve showing the tumor volume of mice inoculated with tumorcells and treated with wild type strains of Neospora caninum-1 (NC-1)compared to untreated mice inoculated with the tumor only (control).

FIG. 5 is a dot plot showing the tumor volume of mice inoculated withtumor cells and treated with wild type strains of Neospora caninum-1(NC-1) compared to untreated mice inoculated with the tumor only(control) at day 25. **: p-value <0.01 (Kruskall Wallis test).

FIG. 6 is a dot plot showing the serum level of IFNgamma before or 4days post-infection (4 DPI) with wild type strains of Neospora caninum-1in mice implanted with the tumor.

FIG. 7 is a dot plot showing the in vitro IFNgamma production by spleenlymphocytes recovered from mice treated with wild type strains ofNeospora caninum-1 (NC-1) or untreated (Control) and stimulated withconcanavalin A (ConA).

FIG. 8 is an immunoblot showing GRA15II protein expression (visualizedby anti-Tag HA). The black arrow indicates the GRA15II protein expectedband at 60 kDa molecular weight. Lane 1: Neospora caninum parasiteselectroporated with a plasmid encoding GRA15II. Lane 2: untransducedNeospora caninum parasites. Lane 3: molecular weights (MW) standards:17, 26, 42, 55, 72, 95 and 140 kDa.

EXAMPLES Example 1: In Vivo Effect of Neospora caninum Materials AndMethods

Mice

Eight week-old female inbred C57BL/6 mice are maintained underpathogen-free conditions.

Neospora caninum-1 Strain

Tachyzoites of the NC-1 strain of Neospora caninum are harvested frominfected human foreskin fibroblasts Hs 27 (ATCC CRL-1634) cultured inmonolayers in DMEM, supplemented with 10% heat-inactivated FCS, 50 U/mlpenicillin/50 μg/ml streptomycin, and 1% HEPES. Neospora caninumtachyzoites may be harvested when monolayers of human foreskinfibroblasts are completely lysed.

Tumor Cells

EG7 cells (EL4-OVA thymoma cells transfected with chicken albumin cDNA)are cultured, for example, in RPMI medium, with 5×10⁵ M of2-mercaptoethanol, 50 UI/mL of penicillin and 50 mg/mL of streptomycin.

Tumor Cell Inoculations 1×10⁵ live EG7 cells are inoculatedintradermally in the right flank of the mice. Tumor diameters aremeasured 3 times weekly, and mice are euthanized when tumor diametersreached 25.000 mm³.

Neospora caninum-1 (NC-1) Administration

Mice are injected subcutaneously in the right flank at day 4 and againat day 7 with 5×10⁶ freshly isolated tachyzoites of NC-1 strain ofNeospora caninum.

Cell Culture Conditions and Cytokine Quantification

Spleens are harvested and pressed through a stainless steel mesh. Singlecell suspensions are obtained by filtration through a nylon mesh toremove tissue debris.

Spleen erythrocytes are lysed by hypotonic shock, single cells of spleenare resuspended in RPMI 1640 supplemented with 5% FCS, HEPES (25 mM),L-glutamine (2 mM), sodium pyruvate (1 mM), β-mercaptoethanol (5×10⁻⁵ M)and penicillin (50 U/ml)/streptomycin (50 μg/ml). Spleen cells arecultured in 96-well plates at 5×10⁵ cells per well, in 200 μl of culturemedium, alone or containing concanavalin A (10 μg/ml). The plates areincubated for 72 hours in 5% CO2 at 37° C. Cell culture supernatants areharvested and kept at −20° C. IFN-gamma quantification is determined byan ELISA assay on the supernatant of the spleen lymphocytes.

Results

Neospora caninum Infection is Lethal at Very High Inoculum Doses

Strains of Neospora caninum challenging study reveals that all miceimmunized with 5×10⁶, 5×10⁵, 5×10⁴, and 5×10³ of NC-1 tachyzoitessurvive up to 25 days. One on three mice infected with 5×10′ NC−1tachyzoites succumbs on day 5 (FIG. 1).

Neospora caninum Induces Humoral and Cellular Immune Responses

High levels of seric IFN-gamma (approximatively 2000 pg/mL) are obtainedat day 8 post-infection whatever the doses of injected NC-1 tachyzoites.IFN-gamma secretion at day 2 post-infection is only observed in serum ofmice infected with 5×10⁷ and 5×10⁶ NC-1 tachyzoites (FIG. 2).

All infected mice develop Neospora-specific humoral immune responses atday 21 post-infection. As shown in FIG. 3, highest levels of humoralantibodies are achieved in mice infected with 5×10⁷ and 5×10⁶ NC-1tachyzoites (approximatively 0.8 for a dilution 1/50) (FIG. 3).

According to these results showing a correlation between the dose andthe anti-Neospora immune response, it was decided to focus on theconcentration of 5×10⁶ NC-1 tachyzoites for the next experiments.

Neospora caninum Treatment Suppresses and/or Regresses an EstablishedSolid Tumor Development

Mice that received EG7 cells develop large tumors (8090±507 mm³) at day25 post transfer. In contrast, pretreatment with NC-1 tachyzoitessignificantly reduces the tumor volume (1816±465 mm³), demonstratingthat NC-1 tachyzoites suppress the thymome tumor development (FIGS. 4and 5).

Neospora caninum Induces a Protective Immune Response Against TumorDevelopment

An increase of serum level of IFN-gamma 8 days post tumor implantationand 4 days after the first Neospora dose inoculation (4 DPI) is observedin Neospora-treated mice compared to serum level of IFN-gamma inuntreated mice (FIG. 6).

An increase of IFN-gamma production is observed in vitro by spleenlymphocytes recovered from Neospora-treated mice (NC-1) and stimulatedwith concanavalin A compared to stimulated spleen lymphocytes recoveredfrom untreated mice (control). This result suggests that Neosporacaninum generates a therapeutic antitumor immune response by reversingspleen immunosuppression during tumor development (FIG. 7).

Example 2: Neospora caninum Over-Expressing GRA15II

In order to obtain a Neospora caninum strain overexpressing GRA15, thesequence encoding GRA15II (strain Me49, sequence ToxoDB, Gene ID:7895856, TGME49_275470) including a HA-Tag at the C-terminal end wascloned in a vector pUC8 at the Pmel site. The vector pUC8 contains 2expression cassettes. One expression cassette encodes thechloramphenicol resistance gene in fusion with the GFP protein under thecontrol of the pTUB5 promoter. The second expression cassette encodesthe protein of interest, GRA15II, under the control of the pTUB8promoter whose sequence includes the Kozak sequence, the translationinitiation codon and the stop codon.

Neospora caninum NC-1 parasites (10⁷ parasites), taken up in Cytomixmedium (120 mM KCl; 5 mM MgCl₂; Hepes 25 mM; 10 mM K₂HPO4/KH₂PO4, pH7.6; 2 mM EDTA; 0.15 mM CaCl₂, pH 7.6 adjusted with KOH), containing 3mM ATP and 3 mM Glutathione were then electroporated with 50 μg ofrecombinant plasmid pUC8 GRA15II previously linearized with the Pcilrestriction enzyme.

Then, parasites cultured on Human Foreskin Fibroblasts (HFF cells) wereselected with a medium containing chloramphenicol. After eight weeks ofselection, the chloramphenicol-resistant parasites were analyzed byimmunoblotting to visualize the expression of the GRA15II protein.

The expression of GRA15II protein was analyzed by electrophoresis (5.10⁶parasites per well) followed by a transfer on nitrocellulose membrane.Briefly, parasites were suspended in sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer,sonicated, heated at 100° C. for 3 mM, and separated on a polyacrylamidegel. After electrophoresis, proteins were transferred onto anitrocellulose membrane, which was probed with anti-HA Tag PolyclonalAntibody (71-5500—Invitrogen).

Bound antibodies were detected using anti-rabbit immunoglobulin G (IgG,whole molecule)—alkaline phosphatase conjugate (A3687—Sigma-Aldrich).Alkaline phosphatase activity was detected using the5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT)liquid substrate system (S3771—Promega). Molecular masses standards(prestained SDS-PAGE standards, (ProSieve QuadColor Protein Markers,Lon00193837—Ozyme)) were used.

The protein is expected at 60 Kda molecular weight. A band indicated bythe black arrow is visualized in Lane 1 at approximately 60 Kda (FIG. 8)showing the GRA15II protein expression in Neospora caninumelectroporated with the PUC8 GRA15II plasmid in comparison withnon-electroporated Neospora caninum in Lane 2 (FIG. 8) showing no bandat 60 kda. Bands visualized above the indicated MW may correspond to adimerized form of the GRA15II protein.

The invention claimed is:
 1. A method for reducing a tumor size in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one strain of Neospora caninum, wherein said at least one strain of Neospora caninum is at a tachyzoite stage.
 2. The method according to claim 1, wherein said at least one strain of Neospora caninum at a tachyzoite stage is a wild type strain.
 3. The method according to claim 1, wherein said at least one strain of Neospora caninum at a tachyzoite stage is a mutant strain characterized by an over-expression of GRA15 protein, and/or by an under-expression of ROP16 protein.
 4. The method according to claim 1, wherein said tumor is a solid tumor.
 5. The method according to claim 1, wherein said tumor is an ovarian cancer, pancreatic cancer or melanoma.
 6. The method according to claim 1, wherein said at least one strain of Neospora caninum at a tachyzoite stage is comprised in a composition in association with an excipient.
 7. The method according to claim 1, wherein said at least one strain of Neospora caninum at a tachyzoite stage is comprised in a pharmaceutical composition further comprising at least one pharmaceutically acceptable excipient.
 8. The method according to claim 1, wherein said at least one strain of Neospora caninum at a tachyzoite stage is comprised in a vaccine composition.
 9. The method according to claim 1, wherein said at least one strain of Neospora caninum at a tachyzoite stage is comprised in a vaccine composition comprising an adjuvant.
 10. The method according to claim 1, wherein said at least one strain of Neospora caninum at a tachyzoite stage is administered to the subject via subcutaneous, intradermal or intratumoral routes.
 11. The method according to claim 1, wherein the therapeutically effective amount of Neospora caninum at a tachyzoite stage administered to the subject is ranging from about 10⁴ to about 10¹¹ cells. 